Methods of purifying recombinant alpha-Galactosidase A

ABSTRACT

In one embodiment, the invention provides a method of purifying recombinant alpha-galactosidase A. The method includes obtaining a lysate from cells recombinantly expressing alpha-galactosidase A grown in a cell culture medium having non-precipitating phosphate; contacting said lysate with a first chromatography media that binds α-D-mannopyranosyl or α-D-glucopyranosyl; eluting alpha-galactosidase A from the first chromatography media to generate a first eluate having alpha-galactosidase A, wherein said eluting includes at least one elution pause between 4 and 16 hours; contacting the first eluate with a second chromatography media that binds galactose binding proteins; and eluting alpha-galactosidase A from said second chromatography media to generate a second eluate containing said recombinant alpha-galactosidase A.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of application Ser. No.14/732,662, filed Jun. 5, 2015, which claims priority from U.S.Provisional Application No. 62/011,399, filed on Jun. 12, 2014, all ofwhich is incorporated herein by reference.

INCORPORATION OF SEQUENCE LISTING

Incorporated herein by reference in its entirety is the Sequence Listingfor the application. The Sequence Listing is disclosed on acomputer-readable ASCII text file titled,“Sequence_Listing_1038-142CON.txt”, created on Oct. 16, 2020. Thesequence_listing.txt file is 1.3 KB in size.

This invention was made with government support under grant numberNIH/NCRR/RCMI Grant G12-RR03060 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Mutations in the αGal gene result in the sphingolipidosis named Fabrydisease [1]. The enzymatic defect is inherited as an X-linked disorderand is associated with a progressive deposition of theglycosphingolipids, including globotriaosylceramide,galabioasylceramide, and blood group B substance. In affected males thisleads to early death due to occlusive disease of the heart, kidney, andbrain.

De Duve [2] first suggested that ERT might be a successful approach tothe treatment of lysosomal storage defects such as Gaucher's and Fabrydisease. For Gaucher's disease, ERT produced unequivocal clinicalresponses [3, 4] that were subsequently confirmed by others [5-7].Classical Fabry disease patients lack detectable levels of αGal [1] soit should not be surprising that more than 80% of Fabry patients treatedwith agalsidase-beta [8] and more than 50% treated with agalsidase-alfa[9] developed an immune response. The antibodies produced are primarilyof the IgG class and a fraction of the antibodies appear to exhibitneutralizing properties. These antibodies have been associated with anincrease in urinary globotriaosylceramide levels due to the uptake ofimmune-enzyme complexes by granulocytes in the bloodstream andmacrophages in the tissues [10-12].

ERT for Fabry disease patients was initially undertaken for males withthe classic form of the disease (no detectable αGal activity) in avariety of clinical trials [8, 9, 13-16], but therapy is now alsounderway for heterozygous females with Fabry disease [17-19] and isunder consideration for children [20-22] and adults with atypical (lowlevels of enzyme) Fabry disease [23]. The two products used for ERT inFabry disease patients have been compared [24]. The pattern ofglycosylation on αGal has been analyzed [25] and its importance foractivity [26] and uptake by cells has been established [27, 28]. Thelimitations of current approaches for ERT for Fabry disease and the needfor improved techniques have been discussed [10, 29, 30]. Efforts forgene therapy for Fabry disease are underway [31-38] and molecularchaperones are under investigation for specific alleles [39-41].Substrate reduction therapy as an augmentation to ERT has been evaluated[42]. There are several reviews on the general topic of ERT forlysosomal storage diseases [43-47].

Expression of the human αGal has been reported in Escherichia coli [48],baculovirus [49, 50] Chinese hamster ovary cells [51] and human foreskinfibroblasts [52]. The highest levels of heterologous αGal expressionwere observed in Pichia pastoris [53]. Recombinant αGal has also beenproduced in a modified strain of Saccharomyces cerevisiae thatsynthesized glycoprotein lacking the outer chain of N-glycan, astructure that is specific to yeast but not humans [28, 54]. When thisαGal was introduced into Fabry patient fibroblasts or a Fabry mousemodel, there was hydrolysis of accumulated substrates [28, 54]. Themethylotrophic yeast P. pastoris is the most highly developed of a smallgroup of alternative yeast species chosen for their advantages over S.cerevisiae as expression hosts [55, 56]. Two attributes critical in itsselection are the existence of well-established fermentation methods andthe presence of the tightly regulated methanol-inducible promoter. AOXexpression is undetectable by enzyme assay or mRNA production in cellscultured on carbon sources such as glycerol, but constitutes up to 30%of total soluble protein in methanol-grown cells. Heterologous genesunder the control of the PAOX1 promoter can be maintained in anexpression-off mode on a non-methanolic carbon source in order tominimize expression of potentially toxic heterologous proteins duringcell growth. The P. pastoris expression system has now been successfullyused to produce a number of heterologous proteins at commercially usefulconcentrations [57].

Lysosomal enzymes such as αGal are glycoproteins that are modified inthe Golgi to contain N- or O-linked carbohydrate structures [58]. Thehuman αGal is glycosylated at Asp residues 139, 193, and 215 [26] withbranched carbohydrate structures that vary in composition and sequencedepending upon the host species and tissue type [25]. For example, theenzyme purified from humans contains variable amounts (5-15%) ofasparagine linked complex and high mannose oligosaccharide chains [1].Consequently, multiple forms are present in SDS gels and in isoelectricfocusing experiments that correspond to the plasma and various tissueforms. The Carboxyl-Terminal Truncations of the Human α-Galactosidase Arecombinant human αGal preparations used therapeutically are produced inhuman and CHO cells and these have distinct glycosylation patterns anddiffer in levels of sialic acid and mannose-6-phosphate [24]. Therecombinant αGal produced in insect cells [49, 50] and in P. pastoris[53] contain variable levels of mostly complex and high mannose sidechains, respectively. Glycoproteins produced in P. pastoris typicallycontain from 6 to 14 mannose units (Man6-GlcNac2 to Man14GlcNac2) thatsometimes produces a Gaussian-like distribution of oligomannosides thatmay center near Man12GlcNac2 to Man13GlcNac2 [59].

These carbohydrate moieties serve a structural and functional role. Forexample, it has been demonstrated that glycosylation, particularly atAsn-215, is required for enzyme solubility [26]. Also, uptake of theenzyme by cells in vivo is affected by terminal mannose-6-phosphateresidues on the enzyme [27], and the 10-12 sialic acid residues on theplasma form of the enzyme accounts for the prolonged circulatoryhalf-life of the enzyme compared to the tissue form with only one or twosialic acid residues [60]. The identification of these multiple forms asderivatives of the same protein in purified enzyme preparations canconveniently be monitored by treatment with specific N-glycosidases orby Western blots.

Fabry disease patients with adverse reactions to the infusions arecurrently treated with antihistamines and antipyretics and the initialimmune response has been manageable to date [61, 62], but it can beanticipated that life-long treatment required for these patients willlead to unacceptable levels of neutralizing antibodies. In this contextit is reasonable to devise approaches to circumvent these adversereactions and the development of derivatives of the enzyme with moreactivity per mg is a logical approach. Miyamura and coworkers [63]reported that carboxyl-terminal deletions of 2 to 10 amino acids of αGalled to an increase in activity of about 4 to 6-fold as compared to wildtype (WT). However, this data was qualitative or semi-quantitative andrelied on comparison of the amounts of mRNA present in Northern blots toαGal enzyme activity during transient infection of COS-1 cells. Here weuse a P. pastoris expression system for the construction andpurification of mutant enzymes with C-terminal deletions. Thequantitative results reported here with purified enzymes reveal thatC-terminal deletions results in an increase (Δ2, Δ4, Δ6, and Δ10) ordecrease (Δ8) in enzyme activity.

Accordingly, there is a need for a method to purify recombinantα-Galactosidase A that provides high yield and maintains enzymaticactivity.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method of purifyingrecombinant alpha-galactosidase A. The method includes obtaining alysate from cells recombinantly expressing alpha-galactosidase A grownin a cell culture medium having non-precipitating phosphate; contactingsaid lysate with a first chromatography media that bindsα-D-mannopyranosyl or α-D-glucopyranosyl; eluting alpha-galactosidase Afrom the first chromatography media to generate a first eluatecontaining alpha-galactosidase A, wherein said eluting includes at leastone elution pause between 4 and 16 hours; contacting the first eluatewith a second chromatography media that binds galactose bindingproteins; and eluting alpha-galactosidase A from said secondchromatography media to generate a second eluate containing saidrecombinant alpha-galactosidase A.

In another embodiment, the invention provides a method of purifyingrecombinant human alpha-galactosidase A. The method includes obtaining alysate from cells recombinantly expressing alpha-galactosidase A grownin a cell culture medium having non-precipitating sodiumhexametaphosphate as a phosphate source; contacting the lysate with afirst chromatography media includes Concanavalin A; elutingalpha-galactosidase A from said first chromatography media to generate afirst eluate containing alpha-galactosidase A, wherein said elutingincludes at least one elution pause of about 10-14 hours; contacting thefirst eluate with a second chromatography media, wherein the secondchromatography media includes D-galactose; and elutingalpha-galactosidase A from said second chromatography media to generatea second eluate containing said recombinant human alpha-galactosidase A.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts introduction of a C-terminal deletion of 2 amino acidsinto αGal. The strategy shown here for the Δ2 mutant was used togenerate all five deletion mutations (FIG. 2). Plasmid pMS118 [48]contains the αGal cDNA cloned as an EcoRI fragment to the EcoRI site ofplasmid pUC9. Primers AAF and AARD2 (FIG. 2) were used as PCR primersfor plasmid pMS118 DNA to generate cDNAs with a 50 extension containingan XhoI site, Kex2 and Ste13 yeast signal cleavage sites, a 30 end withan introduced XbaI site, and a deletion of C-terminal amino acids togenerate the Δ2 mutant. Primer AAF anneals to the cDNA at the sequencesencoding the N-terminal sequences of αGal and primer AARD2 anneals tothe C-terminal sequences of αGal. Primer AARD2 anneals 12 nucleotidesfrom the 30 end of the cDNA and introduces a stop codon (UAA) after theaspartate codon three amino acids from the C-terminal end of the codingsequences of αGal resulting in a deletion of the two C-terminal aminoacids (Leu-Leu) of the human enzyme (right panel). Cloning to the XhoIand XbaI sites of plasmid pPICZαA generates a protein fusion with theyeast signal peptide coding sequences in the vector. This signal peptideis removed by the Kex2 and Ste13 yeast signal peptidases throughcleavage immediately upstream of the leucine, corresponding the firstamino acid of the mature form of αGal (left panel). This strategy wasgeneralized to create the other deletion mutants using the primers inFIG. 2. In the left panel, the N-terminal peptide LDNGLAR was identifiedin mass spectrometric analysis while EALDNGLAR was not (FIG. 5).

FIG. 2 depicts primers and αGal cDNA used to generate Δ2, Δ4, Δ6, Δ8 andΔ10 mutant cDNAs. DNA primers AAF, AARD2, AARD4, AARD6, AARD8 and AARD10corresponding to the Δ2, Δ4, Δ6, Δ8 and Δ10 mutants were annealed to thecDNA of mature αGal contained within pMS118 to generate 30 end truncatedPCR products for carboxy-terminal deleted enzymes. Primer AAF andprimers AARD2 to AARD10 (indicated above) were annealed to the 50 and 30ends of the cDNA, respectively. Primer AAF contains an XhoI site(indicated above) and partially encodes for a yeast signal peptide (seeFIG. 1) to produce a fusion protein targeted for secretion from P.pastoris. Primers AARD2 to AARD10 were used to introduce an XbaI site(indicated above) and a premature UAA stop codon via an antisense ATTtriplet immediately downstream of nucleotides complementary to αGal(bold font) to produce cDNAs encoding for Δ2, Δ4, Δ6, Δ8, Δ10 mutants.The boxed LDNGLAR and SHINPTGTVLLQLENTMQM protein sequences (indicatedabove) are peptide fragments that were identified through massspectrometry of the Δ6 mutant (FIG. 5).

FIG. 3 depicts SDS-PAGE for purification of αGal. Purified samples wererun on a 4-20% polyacrylamide gel, under reducing conditions, andstained with Coomassie Brilliant Blue. The contents of the lanes are asfollows: molecular weight marker (lane 1 and 8), WT(PC626) (lane 2), Δ2(PC995) (lane 3), Δ4 (PC897) (lane 4), Δ6 (PC958) (lane 5), Δ8 (PC973)(lane 6), Δ10 (PC960) (lane 7). The minor bands present in the purifiedfraction are consistent with high molecular weight glycoforms seenpreviously when WT enzyme was purified from the same P. pastorisexpression system [53].

FIGS. 4A-B depict a Western Blot of purified WT and mutant αGal.Purified WT and mutant enzymes were subjected to Western blotting usinga polyclonal antibody raised against residues 55-64 and 396-407 of αGal.FIG. 4A blot at shorter and FIG. 4B longer exposure.

FIGS. 5A-B depict mass spectrometry of purified Δ6 αGal. MS/MS spectraobtained from parental MS ions (FIG. 5A) m/z=379.71 and (FIG. 5B)m/z=1064.03 corresponding to a C-terminal and N-terminal peptide,respectively. Product ion peaks are annotated according to theirpredicted [M+H]+ forms. Annotations in red and blue correspond tob-series and y-series ion fragments, respectively.

FIGS. 6A-B depict thermostability profiles of WT and mutant αGal.Stability of recombinant WT and Δ2 to Δ10 mutant αGal at 30° C. (FIG.6A), 40° C. (FIG. 6B), and 50° C. (FIG. 6C) at pH 5.5 as monitored byfluorescent enzyme assay. Initial activities ranged from approximately300 to 1,900 units/mL for all enzymes assayed. % Activity is normalizedagainst activity at t=0 mins. Data points for (FIG. 6A) and (FIG. 6C)are the mean of a triplicate measurement with error bars equivalent to±1 standard deviation. Data points for (FIG. 6B) are the results of asingle measurement. MUG was used as the substrate for enzyme assay.

FIG. 7 depicts pH activity curves of WT and mutant αGal. pH activitycurves for WT and Δ2 to Δ10 mutant αGal. % Activity is normalizedagainst each enzyme's peak activity. Data points are the mean of atriplicate measurement and error bars are ±1 standard deviation. MUG wasused as the substrate for enzyme assay.

FIGS. 8A-B depict substrate saturation curves of WT and mutant αGal.Purified WT, Δ2, Δ4, Δ6, Δ8 and Δ10 αGal were enzyme assayed in 0.3 to2.0 mM MUG (FIG. 8A) and in 7 mM to 50 mM PNPαGal (FIG. 8B) to measureinitial velocities (mmol product per hr/mg enzyme). Km and Vmaxparameters were extracted and compiled in Table 3. The figure indicatesfits of Michaelis-Menten hyperbolas to experimental data indicated asmean±one standard deviation.

FIGS. 9A-B depict quantification of Bands in SDS-PAGE (FIG. 9A). Bandintensities of the SDS-PAGE in (FIG. 3) were quantified by ImageAcquisition and Analysis software (VisionWorks®LS, UVP Inc., Upland,Calif.) FIG. 9B.

FIGS. 10A-C depict chromatogram (FIG. 10C) and fraction analysis (FIGS.10A and 10B) of binding, washing, and elution sample throughConcanavalin A(ConA) Sepharose 4B column.

FIGS. 11A-B depict the chromatogram (FIG. 11B) and fraction analysis(FIG. 11A) of binding, washing, and elution of sample through a Thiogalcolumn.

FIG. 12 depicts SDS-PAGE of sample fractions obtained from thepreparation shown in FIGS. 10 and 11.

FIG. 13 depicts enzymatic activity of alpha-galactosidase A at differentstages of purification.

DETAILED DESCRIPTION

The invention provides a method of purifying recombinantalpha-galactosidase A. The alpha-galactosidase A may be humanalpha-galactosidase A, non-human alpha-galactosidase A, or a chimericalpha-galactosidase A having similarity to human and non-humansequences. The recombinant alpha-galactosidase A may also have mutationsthat alter the catalytic activity. The recombinant alpha-galactosidase Amay be full length or truncated. Examples of truncatedalpha-galactosidase A include those having C-terminal truncations of 2,4, 6, 8, 10, 12, or 14 amino acids.

A lysate is obtained from cells recombinantly expressingalpha-galactosidase A grown in a cell culture medium havingnon-precipitating phosphate. Any suitable cell capable of stablyexpressing alpha-galactosidase A may be used. An example of a suitablecell includes Pichia pastoris. Any suitable expression system capable ofexpressing alpha-galactosidase A may be used. Expression systems includeplasmid borne expression systems or expression systems integrated withinthe genome. In some embodiments, non-precipitating sodiumhexametaphosphate is the phosphate source for the culture medium. Inother embodiments, phosphate glass may be used as the phosphate source.Sodium trimetaphosphate and/or sodium orthophosphate have also beencontemplated as the phosphate source. The lysate may be generated by anyknown means. For example, cells recombinantly expressingalpha-galactosidase A may be harvested and the cells lysed by mechanicalmeans or chemical means. An example of mechanical lysis includessonication. The lysate may be clarified by at least one ofcentrifugation or filtration. Examples of filtration include passing thelysate through a 0.2 μm hollow fiber filter, and diafiltration across a50 kDa pore size hollow fiber filter.

The lysate is then contacted with a first chromatography media to bindalpha-galactosidase A to the first chromatography media. As used herein,chromatography media may be in the form of a packed column orfree-flowing slurry. The first chromatography media includes any mediathat binds α-D-mannopyranosyl, or α-D-glucopyranosyl. Examples of suchmedia include Concanavalin A (ConA) immobilized on Sepharose™ 4B; andmannose-specific lectin from the snowdrop (Galanthus nivalus) bulbimmobilized on a solid matrix. The first chromatography media may thenbe washed to remove any material non-specifically bound to it.

The alpha-galactosidase A bound to the first chromatography media iseluted by contacting the first chromatography media with an elutionbuffer to generate an eluate containing alpha-galactosidase A. Eluate,as used herein, is defined as elution buffer that has contacted thechromatography media and contains alpha-galactosidase A. This eluate isthe first eluate. Any elution buffer capable of disrupting the bindingof alpha-galactosidase A to the first chromatography media may be used.For example, the elution buffer may contain an increasing gradient(linear or step) of α-D-methylmannoside or α-D-methylglucoside. The useof glucose and mannose has also been contemplated. The elution buffermay also have a pH less than 6, less than 5, less than 4.5, or less than4. The elution buffer contains non-near-saturating concentration ofsugar.

Elution of alpha-galactosidase A from the first chromatography media mayinclude at least one elution pause. An elution pause, as used herein, isdefined as contacting elution buffer with the chromatography media, andpausing the flow of the elution buffer such that there is no flow acrossthe chromatography media. By way of further explanation, thechromatography media is incubated with the elution buffer. In the caseof column chromatography, for example, one or more column volumes ofelution buffer are flowed through the chromatography column, and theflow is paused. In the case of batch chromatography, a defined volume ofelution buffer is contacted with the media and maintained for an amountof time (pause). In one embodiment, the pause may be from 4-16 hours. Inanother embodiment, the pause is from 12-14 hours. In one embodiment,the flow is paused for at least 4 hours, preferably for at least 8hours, and most preferably at least 12 hours.

The first eluate is contacted with a second chromatography media to bindalpha-galactosidase A to the second chromatography media. The firsteluate may be subject to diafiltration, dialysis, or buffer exchangeprior to contacting the second chromatography media. The secondchromatography media is any media that binds galactose binding proteins.Examples of such media include D-galactose or C-type lectins. C-typelectin mutants engineered to have increased affinity for galactose havealso been contemplated.

The alpha-galactosidase A is eluted from said second chromatographymedia by contacting it with a second elution buffer. Any elution buffercapable of disrupting the binding of alpha-galactosidase A to the secondchromatography media may be used. The second elution buffer may containan increasing gradient (linear or step) of D-galactose. The elutionbuffer may also have a pH less than 6, less than 5, less than 4.5, orless than 4.

In the specification, numerous specific details are set forth in orderto provide a thorough understanding of the present embodiments. It willbe apparent, however, to one having ordinary skill in the art that thespecific detail need not be employed to practice the presentembodiments. In other instances, well-known materials or methods havenot been described in detail in order to avoid obscuring the presentembodiments.

Throughout this specification, quantities are defined by ranges, and bylower and upper boundaries of ranges. Each lower boundary can becombined with each upper boundary to define a range. The lower and upperboundaries should each be taken as a separate element.

Reference throughout this specification to “one embodiment,” “anembodiment,” “one example,” or “an example” means that a particularfeature, structure or characteristic described in connection with theembodiment or example is included in at least one embodiment of thepresent embodiments. Thus, appearances of the phrases “in oneembodiment,” “in an embodiment,” “one example,” or “an example” invarious places throughout this specification are not necessarily allreferring to the same embodiment or example. Furthermore, the particularfeatures, structures or characteristics may be combined in any suitablecombinations and/or sub-combinations in one or more embodiments orexamples. In addition, it is appreciated that the figures providedherewith are for explanation purposes to persons ordinarily skilled inthe art and that the drawings are not necessarily drawn to scale.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,article, or apparatus that comprises a list of elements is notnecessarily limited to only those elements but may include otherelements not expressly listed or inherent to such process, article, orapparatus.

Further, unless expressly stated to the contrary, “or” refers to aninclusive “or” and not to an exclusive “or”. For example, a condition Aor B is satisfied by any one of the following: A is true (or present)and B is false (or not present), A is false (or not present) and B istrue (or present), and both A and B are true (or present).

Additionally, any examples or illustrations given herein are not to beregarded in any way as restrictions on, limits to, or expressdefinitions of any term or terms with which they are utilized. Instead,these examples or illustrations are to be regarded as being describedwith respect to one particular embodiment and as being illustrativeonly. Those of ordinary skill in the art will appreciate that any termor terms with which these examples or illustrations are utilized willencompass other embodiments which may or may not be given therewith orelsewhere in the specification and all such embodiments are intended tobe included within the scope of that term or terms. Language designatingsuch nonlimiting examples and illustrations includes, but is not limitedto: “for example,” “for instance,” “e.g.,” and “in one embodiment.”

In this specification, groups of various parameters containing multiplemembers are described. Within a group of parameters, each member may becombined with any one or more of the other members to make additionalsub-groups. For example, if the members of a group are a, b, c, d, ande, additional sub-groups specifically contemplated include any one, two,three, or four of the members, e.g., a and c; a, d, and e; b, c, d, ande; etc.

EXAMPLES

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1. Materials and Methods

Cell Strains and Plasmids

The P. pastoris host strain X-33 (No. K1740-01), E. coli strains TOP10(No. C4040-50) and TOP10F′ (No. C665-11), plasmid pPICZαA (No.K1740-01), and TOPO® XL PCR cloning kit (No. K4700-10) were purchasedfrom Invitrogen.

TABLE 1 Table 1. Strains and Plasmids. Strain Species PlasmidDescription CC878 E. coli pCC248 pCR-XL-TOPO derivative plasmid amodified cDNA using primers AAF and AARD4 to generate C-terminaldeletion of 4 amino acids (Δ4) CC892 E. coli pCC262 pPICZαA derivativeplasmid with Δ4 CDNA insert CC970 E. coli pCC278 pCR-XL-TOPO derivativeplasmid a modified cDNA using primers AAF and AARD6 to generateC-terminal deletion of 6 amino acids (Δ6) CC973 E. coli pCC281pCR-XL-TOPO derivative plasmid a modified cDNA using primers AAF andAARD10 to generate C-terminal deletion of 10 amino acids (Δ10) CC983 E.coli pCC291 pPICZαA derivative plasmid with Δ6 CDNA insert CC990 E. colipCC298 pPICZαA derivative plasmid with Δ10 CDNA insert CC993 E. colipCC301 pCR-XL-TOPO derivative plasmid a modified cDNA using primers AAFand AARD2 to generate C-terminal deletion of 2 amino acids (Δ2) CC994 E.coli pCC302 pCR-XL-TOPO derivative plasmid a modified cDNA using primersAAF and AARD8 to generate C-terminal deletion of 8 amino acids (Δ8)CC995 E. coli pCC303 pPICZαA derivative plasmid with Δ2 cDNA insertCC997 E. coli pCC305 pPICZαA derivative plasmid with Δ8 cDNA insertPC626 P. pastoris pCC106 Integrated pPICZαA derivative with WT cDNAinsert [53] PC897 P. pastoris pCC262 Integrated pPICZαA derivative withΔ4 cDNA insert PC958 P. pastoris pCC291 Integrated pPICZαA derivativewith Δ6 cDNA insert PC960 P. pastoris pCC298 Integrated pPICZαAderivative with Δ10 cDNA insert PC971 P. pastoris pCC303 IntegratedpPICZαA derivative with Δ2 cDNA insert PC973 P. pastoris pCC305Integrated pPICZαA derivative with Δ8 cDNA insert TOP10 E. coli None E.coli host for modified Δ2, Δ6, Δ8, Δ10 cDNA plasmids TOP10F′ E. coliNone E. coli host for pCC106 and modified Δ4 cDNA plasmid X-33 P.pastoris None Expression host for αGal expression Doi:10.1371/journal.pone.0118341.t001

TABLE 2 Primers Used for DNA Sequence Analysis. Primer Sequence Function5′ AOX 5′ GACTGGTTCCAATTGACAAGC 3′ DNA sequencing primer for pPICZαA(SEQ ID NO: 1) 3′ AOX 5′ GCAAATGGCATTCTGACATCC 3′DNA sequencing primer for pPICZαA (SEQ ID NO: 2) α-factor5′ TACTATTGCCAGCATTGCTGC 3′ DNA sequencing primer for pPICZαA(SEQ ID NO: 3) M13: forward 5′ GTAAAACGACGGCCAG 3′DNA sequencing primer for pCR-XL-TOPO (SEQ ID NO: 4) M13: reverse5′ CAGGAAACAGCTATGAC 3′ DNA sequencing primer for pCR-XL-TOPO(SEQ ID NO: 5) Note. Primers were HPLC purified, 50 nmoles fromInvitrogen Doi:10.1371/journal.pone.0118341.t002Bioreactor Expression of Recombinant αGal in P. pastoris

High-cell-density fermentation was carried out as previously described[53] with a modified growth medium utilizing non-precipitating sodiumhexametaphosphate as a phosphate source [64] and modified for a 7 LApplikon bioreactor. Fermentation medium of 3.5 L (0.93 g/l CaSO4, 18.2g/l K2SO4, 14.9 g/l MgSO4.7 H2O, 9 g/l (NH4)2SO4, 40.0 g/l glycerol) wasautoclaved at 121° C. for 20 min in the vessel. After cooling to roomtemperature, filter sterilized sodium hexametaphosphate (25 g/l offermentation basal salt medium dissolved in 500 ml of deionized water)and 0.435% PTM1 trace elements (CuSO4.5 H2O 6.0 g, NaI 0.08 g, MgSO4.H2O3.0 g, Na2MoO4.2 H2O 0.2 g, H3BO3 0.02 g, CoC12 0.5 g, ZnCl2 20.0 g,FeSO4.7 H2O 65.0 g, biotin 0.2 g, 5.0 ml H2SO4 per liter) were added tocomplete the fermentation medium. The pH was adjusted to 6.0 usingammonium hydroxide (28%).

Four frozen MGY cultures of 4 ml each were used to inoculate four 100 mlMGY cultures in 1-liter baffled flasks and grown at 250 rpm and 30° C.until the OD600 reached 2 to 6. The cultivation was divided into threephases, the glycerol batch, glycerol-fed batch, and methanol-fed batch.The glycerol batch phase was initiated with 400 ml of inoculumshake-flask culture added to 4 L of the fermentation medium containing4% glycerol and an initial value of 100% dissolved oxygen until a spikewas observed indicating complete consumption of glycerol. Next, theglycerol-fed batch phase was initiated and a 50% w/v glycerol feed rateof 18.15 ml/h/liter initial fermentation volume and maintained until acell yield of 180 to 220 g/liter wet cells was achieved. At this pointthe glycerol feed was terminated manually and a methanol-fed batch phasewas initiated by starting a 100% methanol feed containing 12 ml PTM1trace salts per liter. Methanol was initially fed at 3.6 ml/h/liter ofinitial fermentation volume, then increased to 7.3 ml/h/liter andfinally increased to 10.9 ml/h/liter of initial fermentation volume forthe remainder of the fermentation. Dissolved oxygen spikes were usedduring the glycerol-fed batch phase and methanol-fed batch phase and tomonitor substrate levels. A dissolved oxygen level of 40%, pH of 6, andtemperature of 25° C. were maintained by an ADI 1030 regulator. Samplingwas performed at the end of each phase and at least twice daily andanalyzed for cell wet weight and increased αGal activity over time.Cultivation was terminated once a plateau in αGal activity was observed.

Construction of Strains

Plasmid pMS118 [48] contains the αGal cDNA cloned as an EcoRI fragmentto the EcoRI site of plasmid pUC9. PCR primers (FIG. 1, 2) were usedwith plasmid pMS118 DNA and the PCR system (Roche, No. 11732641001)according to the vendor's instructions. This generated cDNAs with a 5′extension containing an XhoI site, Kex2 and Ste13 yeast signal cleavagesites, a 3′ end with an XbaI site, and a deletion of C-terminal aminoacids to generate Δ2 to Δ10 mutants (FIG. 1, 2). The PCR products wereligated to pCR-XL-TOPO to generate Δ2 to Δ10 plasmids (Table 1). Theseplasmids were used for electroporation [53] into E. coli strain TOP10 orTOP10F′ (Table 1).

Purification of αGal Using Double Affinity Chromatography

Purification was as described [53, 65] with minor modifications (below).Bioreactor supernatant was passed through a 0.2 μm hollow fiber filter(Spectrum Labs, No. M22M-300-01N) and subjected to diafiltration using a50 kDa pore size hollow fiber filter (Spectrum Labs, No. M25S-300-01N)against wash buffer (0.1 M sodium acetate buffer, pH 6.0, 0.1 M NaCl, 1mM MgCl2, 1 mM CaCl2, 1 mM MnCl2). The resulting supernatant was appliedto a Con A Sepharose 4B (GE Healthcare No. 17-0440-01) column,pre-equilibrated with wash buffer, and washed with 5 column volumes ofwash buffer. It was observed that near-saturating sugar eluentconcentrations do not improve glycoprotein recovery as compared to lowerconcentrations and that elution phase pauses improve recovery [66]. Inaccordance with these findings, elution of αGal was carried out usingmodified elution buffer I (0.5 M methyl-α-D-mannopyranoside, 0.25 Mmethyl-α-D-glucopyranoside in wash buffer) over 1.5 column volume blocksseparated by 12-hour interval soaks. Elution was discontinued when theabsorbance at 280 nm and enzyme assays showed negligible presence ofprotein and αGal activity. No substantial difference in recovered enzymewas observed between purifications carried out with modified elutionbuffer I versus sugar saturated elution buffer I (data not shown). TheCon A pool was subjected to diafiltration using a 50 kDa pore sizehollow fiber filter (Spectrum Labs, No. M25S-300-01N) against bindingbuffer (25 mM citrate-phosphate buffer, pH 4.8 containing 0.1 M NaCl).

The Con A pool was applied to an immobilized-D-galactose gel column(Thio-Gal, Pierce No. 20372) pre-equilibrated with binding buffer. Thecolumn was washed with 5 column volumes of binding buffer and αGal waseluted with elution buffer II (25 mM citrate-phosphate buffer, pH 5.5,0.1M NaCl, 0.1MD-galactose) over 1.5 column volume blocks separated by12 hour soaks. Fractions were assayed for enzyme activity and proteinconcentration and a peak tube with high specific activity was chosen asthe sample to be used in a substrate saturation curve.

Electrophoresis Analysis

Samples (8 μg) were mixed with an equal volume of reducing sample buffer(Bio-Rad Laemmli sample buffer with 5% β-mercaptoethanol) and heated for5 minutes at 95° C. before loading on a Mini-Protean TGX Precast Gel4-20% (w/v) (Bio-Rad No. 456-1094). Bands were visualized by Coomassieblue staining via the modified Fairbanks protocol [67].

Western Blot Analysis

Western blot analysis was performed using an anti-αGal polyclonalantibody produced in chicken (Pierce/ThermoSci #PA1-9528) andhorseradish peroxidase-conjugated anti-Chicken IgY antibody (Sigma#Δ9046). After SDS-PAGE (2 μg of samples loaded), the gel was incubatedwith a nitrocellulose membrane (Whatman, No. 10402594) for 15 minutes atroom temperature in Transfer Buffer (48 mM Tris, 39 mM glycine, 20%MeOH, pH 9.2) and the proteins were then transferred to thenitrocellulose membrane using a Bio-Rad Trans Blot SD Semi-Dry TransferCell. The membrane was blocked with 8% (w/v) non-fat dried milk in PBST[10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl and 0.2% Tween 20(pH 7.4)] at room temperature for 20 minutes. The membrane was thentreated with primary antibody diluted in a milk/blot solution [1% (w/v)non-fat dried PBST] for 2 h at room temperature with mild shaking. Afterrinsing with PBST solution, the membrane was treated for 1 h at roomtemperature with secondary antibody diluted in the milk/blot solution.Protein bands were visualized on Kodak BioMax XAR film (VWR #1B1651454)with a Konica SRX-101A processor.

Enzyme and Protein Assays

Activity of αGal was assayed using the synthetic substrate,4-methylumbelliferyl-α-D-galactopyranoside (MUG) as described [53] withmodifications to a microtiter plate format (below).

Enzyme activity is measured in units/ml where one unit is defined as theamount of enzyme required to convert 1 nmole of MUG to4-methylumbelliferone in one hour at 37° C. An aliquot of 3 μl was addedto 27 μl of enzyme assay buffer (5 mM MUG in 40 mM sodium acetatebuffer, pH 4.5). This mixture was incubated at 37° C. and 10 μl aliquotswere taken at two time points and added to 290 μl of 0.1M diethylaminein a microtiter plate to stop the reaction. Typically time points werechosen as 1-4 minutes and values that were proportional to time wereconsidered valid. The fluorescence of each sample was measured at anexcitation wavelength of 365 nm and an emission wavelength of 450 nmusing a Tecan Infinite F200 microtiter plate reader. A standard curve of10 μl of 0-0.5 nmol 4-methylumbelliferone dissolved in MeOH in 290 μl of0.1 Mdiethylamine was used to quantitate MUG cleavage at specific timeintervals. Analysis of the effects of MeOH indicated no effect on the4-methylumbelliferone standard curve.

For samples containing higher protein concentrations, the BioRad DCProtein Assay (No. 500-0116) with a standard curve of (0.2-1.5) mg/mlwas used according to the manufacturer's specifications. For dilutesamples of purified αGal, a more sensitive fluorescence-basedfluorescamine assay [68] with a standard curve containing lower proteinconcentrations of (4.0-160) μg/ml was used. Briefly, 150 μl of 0.05Msodium phosphate buffer and 50 μl of 1.08 mM fluorescamine dissolved inacetone were added to an aliquot of 50 μl of the sample and standards,mixed and incubated for 12 minutes. The fluorescence of each sample wasmeasured at an excitation wavelength of 400 nm and an emissionwavelength of 460 nm. Bovine serum albumin (Bio-Rad No. 500-0112) wasused as the standard in both assays. Absorbance and fluorescencemeasurements were conducted on a Tecan Infinite F200 microplate readerusing 96-well plates.

Mass Spectrometry of a Purified Mutant Enzyme

The Δ6 mutant was selected for mass spectrometry analysis conducted atthe Rockefeller University Proteomics Resource Center in collaborationwith M. T. Mark. SDS-PAGE gel slices were washed, de-stained, reducedusing 10 mM dithiothreitol, alkylated using 100 mM iodoacetamide, anddigested using trypsin. Peptides were then extracted from the gel twotimes, dried, and re-suspended in a 5% acetonitrile and 2% formic acidmixture. One third of each sample was loaded onto a C18 PepMap1000micro-precolumn (300 μm I.D., 5 mm length, 5 μm beads, ThermoScientific) at a flow-rate of 5 μl/min, and subsequently onto ananalytical C18 column (75 μm I.D., 3 μm beads, Nikkyo Technos Co.) at aflow rate of 300 nl/min. The gradient was 40 min long in the range 5 to45% B (buffer A was 0.1% formic acid in water, and buffer B was 0.1%formic acid in acetonitrile). Eluted peptides were applied byelectrospray directly into the LTQ-Orbitrap XL mass spectrometer fromThermo Scientific, operating in a 300 to 1800 m/z mass range. Tandemmass spectrometry was performed by collision induced dissociation usingnitrogen as a collision gas. The resulting spectra were analyzed usingMascot and Proteome Discoverer 1.3 (Thermo Scientific) to identify thepeptides in the sample.

Thermostability and pH Optimum of WT and Mutant αGal

Purified enzyme samples were diluted in 25 mM citrate-phosphate buffer,pH 5.5, 0.1 MNaCl, 0.01 MD-galactose. Samples of 50 μl were incubated intriplicate at 50° C., 30° C. and 40° C. Aliquots of 3 μl were removedfor enzyme assays every 15 minutes for two hours. Samples were assayedin 0.02 M citrate buffer, pH 3.0-pH 6.5, containing 2 mM MUG.

Characterization of Kinetic Properties

Substrate saturation curves for αGal have been reported using MUG atconcentrations up to 2 mM, 5 mM, and 10 mM (in the presence of 0.1% BSAand 0.67% EtOH [24]). We noted that under our experimental conditionsMUG is fully soluble at 2 mM, partially soluble at 5 mM, and chemicallyoversaturated at higher concentrations. Other investigators reported theuse of sonication or detergent treatment to increase the solubility ofMUG (e.g., [69]) but we avoided this approach in order to avoidpotential artifacts due to the use of these techniques. Substratesaturation curves using 2 mM and 5 mM MUG as the highest concentrationswere carried out and the kinetic parameters for αGal were calculatedseparately obtaining similar values. The values reported here (Table 3a)were obtained using a substrate saturation curve of 0.3 to 2 mM MUGsince this is the highest concentration that is fully soluble under ourexperimental conditions. The Km and Vmax values were calculated usingLineweaver-Burk and non-linear regression through the program Sigma-Plot(Systat Software, San Jose, Calif.).

Kinetic parameters were also determined using the colorimetricsubstrate, para-nitrophenyl-α-D-galactopyranoside (PNPαGal) [70].Purified enzymes were diluted to approximately 20,000 units/mL asdetermined by fluorescent MUG assay. These diluted samples were thenadded at a proportion of 1:9 citrate-phosphate buffer (0.1 M) containing7-50 mM PNPαGal. Aliquots of 20 μl of the enzymatic reaction wereremoved at 15 minute intervals to terminate the reaction over the courseof an hour and added to 320 μl of borate buffer (pH 9.8) in a microplate[71]. Product formation was monitored by absorbance at 400 nm. Linearreaction velocities were observed for all measurements. A standard curveof 0-150 μMp-nitrophenylate in borate buffer (pH 9.8) [71] was used toquantitate product formation. Km and Vmax parameters were determinedthrough non-linear regression using Sigma-Plot (Systat Software, SanJose, Calif.).

Protein Structure Analysis

The crystal structure of αGal (PDB 1R47) was viewed and analyzed inPyMOL (Delano Scientific). The MSLDKLL and QMSLKDLL peptidescorresponding to the last 7 or 8 C-terminal amino acids of αGal werebuilt in PyRosetta [72] and visualized in PyMOL [73]. Interatomicdistances were measured using the PyMOL wizard distance command.

A homology model of the coffee bean α-galactosidase was generated on thePhyre2 server[74]. The primary sequence of coffee bean α-galactosidase(GenBank No. AAA33022.1) was set as the query. The crystal structure ofrice α-galactosidase (73% sequence identity to coffee α-galactosidase,PDB#1UAS) was set as the template. Superposition of the coffee homologand human crystal structure of αGal (PDB#1R47) was conducted in PyMOL[73]. Primary sequence alignments were carried out in ClustalOmega(EMBL-EBI).

Example 2. Purification of WT and Mutant αGal

The WT and mutant enzymes were obtained from a 7 L bioreactor andpurified (Table 4, FIG. 3) using Con A and Thio-Gal tandem affinitychromatography. This two column purification is simpler and faster thanour previous purification methods that used three [50] or four [49,53]columns and the yield, degree of purity, and final specificactivities were similar for all three methods.

The non-glycosylated form of αGal (41.8 kDa) is purified from cells asmultiple glycosylated species with a predominant band of about 50 kDaand multiple higher molecular weight forms that differ in extent ofglycosylation (FIG. 3; See Introduction). We have previouslydemonstrated that high molecular weight glycoforms produced in insectcells and P. pastoris can be identified as derivatives of αGal ratherthan contaminants and these glycoforms are converted to a single band onSDS gels of about 41.8 kDa with endoglycosidase treatment [49, 50, 53].In this report we also use a Western blot (FIG. 4) to confirm that thehigh molecular weight forms seen on SDS gels for the WT and deletionmutants are all glycoforms of αGal. In some cases lower molecular weightspecies present in purified enzyme preparations can be identified asαGal fragments in Western blots (e.g., FIG. 4, lane 2). We quantitatedthe distribution of glycoforms in (FIG. 3, FIG. 9) and there is noobvious correlation between the glycosylation pattern and catalyticactivity. It is well established that glycosylation affects enzymestability and enzyme uptake (above) but to our knowledge there is noevidence that the glycosylation pattern affects the catalytic propertiesof this enzyme.

Example 3. Mass Spectrometry of a Purified Mutant Enzyme

Two possible amino terminal amino acids, glutamate or leucine, could beproduced in P. pastoris depending upon the selection of the signalpeptidase, Kex2 or Ste13 (FIG. 1). Due to the fact that potentialimproper amino terminal processing may have an effect on kinetics, weselected one of the purified mutant enzymes (Δ6) for mass spectrometryanalysis in order to identify the amino terminal sequence of thisenzyme. This analysis also made it possible to provide independentverification of the expected changes in the C-terminal amino acidsequence predicted by in vitro mutagenesis (FIG. 1, 2).

The mature form of the enzyme (signal peptide removed; [75]) produced inhumans begins with a leucine codon (FIG. 1, 2). Therefore, tandem massspectrometry following tryptic digestion of the Δ6 αGal purified from P.pastoris could produce tryptic peptides EALDNGLAR or LDNGLAR, dependingupon the use of the Kex2 or Ste13 protease sites (FIG. 1, 2). A putativeLDNGLAR peak was identified in the MS spectra with an m/z of 379.71,consistent with the (M+2H)2+ state of this peptide, while no peaksconsistent with an EALDNGLAR peptide were found. We cannot eliminatewhat we consider to be the less likely possibility that the failure todetect the EALDNGLAR peptide may be due to the failure of the peptide toionize in this MS experiment. Further fragmentation of the m/z=379.71associated peptide peak produced an MS/MS spectrum containing 4 of 7possible y-ions and 4 of 7 possible b-ions from the expectedfragmentation pattern of a hypothetical LDNGLAR peptide (FIG. 5a ). Thisresult indicates that the Ste13 signal peptidase of P. pastorisgenerates an enzyme with an amino terminus identical to the enzymeproduced in humans.

A search for a Δ6 C-terminal tryptic peptide of SHINPTGTVLLQLENTMQM(FIG. 2) yielded a matching MS m/z=1064.03 peak consistent with its(M+2H)2+ state. Further fragmentation also produced an MS/MS spectrumcontaining 4 of 19 possible y-ions and 9 of 19 possible b-ionsconsistent with the anticipated sequence (FIG. 5b ). This resultconfirms the predicted C-terminal deletion of 6 amino acids and confirmsthe efficacy of the mutagenesis protocol used to produce this mutantenzyme.

Thus, the purified Δ6 αGal mutant possesses an N-terminal sequencecorresponding to the mature form of αGal and a C-terminal sequencetruncated by six amino acids.

Example 4. Thermostability and pH Optima of WT and Deletion Mutants ofαGal

Preparations of purified WT and mutant αGal show similar thermostabilityprofiles at 30° C., 40° C., and 50° C., with activity half-lives of 30,25 and 17 minutes, respectively (FIG. 6). The general trend of theseprofiles agree with previous results [76]. All enzymes show optimalactivity near pH 4.5 (FIG. 7) in accord with previous reports for WTαGal [70, 77-79], and there is no significant difference in the activityoptima of purified WT and mutant αGal.

Example 5. Kinetic Analysis of WT and C-Terminal Deletion Mutants

The values for Km and Vmax for WT enzyme (Table 3a) are in accord withpublished values (Table 5). The range of Km and Vmax values for theenzymes purified from several sources in various laboratories over aperiod of more than 30 years (Table 5) are in good agreement and theobserved subtle variations are in the range expected. However, moreprecision is expected for measurements recorded for enzymes purifiedfrom the same source in a single laboratory at one given time (Table3a). Substrate saturation curves (FIG. 8a ) and the calculated valuesfor Km, Vmax, kcat, and kcat/Km using the MUG substrate (Table 3a)reveal differences in the enzyme activity of the mutants compared to WT.Deletions of 2, 4, 6 and 10 amino acids approximately double the kcat/Km(0.8 to 1.7-fold effect; 29/34.4=0.8 and 58.3/34.4=1.7) while a deletionof 8 amino acids decreases the kcat/Km (7.2-fold effect; 34.4/4.78=7.2).There are corresponding changes in the Vmax values and deletions of 2,4, 6 and 10 amino acids approximately double the Vmax (1.5 to 2.2-foldeffect; 4.89/3.36=1.5 and 7.29/3.36=2.2) while a deletion of 8 aminoacids decreases the Vmax (4.5-fold effect; 3.36/0.742=4.5). There arealso smaller differences in the Km values of the C-terminal deletionmutants compared to the WT (Table 3a). The Vmax data presented for theΔ8 (0.742±0.21) and WT (3.36±0.29) are derived from multiple assays fromthree and two independent enzyme samples, respectively, and thisindicates the reliability of this data and adds strength to theinterpretations of the data from the single enzyme preparations used forthe other deletion mutants.

Without wishing to be bound by theory, the effects of the C-terminaldeletions on the kinetic properties of the enzyme using the artificialsubstrate MUG (Table 3a) could be due to alterations in the inherentcatalytic mechanism of the enzyme [85]. Alternatively, the alteredkinetic properties could be due to changes in the affinity of the enzymefor specific structural components of the artificial substrate, MUG. Inthis context, it is of interest to measure these kinetic parameters withan alternative substrate such as PNPαGal. The results (Table 3b, FIG. 8b) indicate that there are similar changes in kinetic parameters usingPNPαGal as the substrate, including increases (2.2-fold effect;9.18/4.18=2.2) and decreases (3.2-fold effect; 4.18/1.31=3.2) in thekcat/Km for the specific C-terminal deletion mutants (Δ10 and Δ8,respectively). Taken together, these results suggest that the C-terminaldeletions likely affect some aspect of the inherent catalytic mechanismof the enzyme.

TABLE 3 Table 3. Values of K_(m), V_(max), k_(cat) and the specificityconstant (k_(cat)/K_(m)) for WT and C-Terminal Deletion Mutants of αGal.K_(m) V_(max) k_(cat) k_(cat)/K_(m) Comments (mM) (mmole/hr/mg) (s⁻¹)(mM⁻¹s⁻¹) A) MUG WT 2.44 ± 0.44 3.36 ± 0.29 84.0 34.4 Δ2 4.52 ± 0.625.56 ± 0.73 139 30.8 Δ4 3.51 ± 0.29 7.29 ± 0.74 182 51.9 Δ6 4.21 ± 0.524.89 ± 0.32 122 29.0 Δ8 3.89 ± 0.27 0.742 ± 0.21  18.6 4.78 Δ10 2.96 ±0.29 6.90 ± 0.71 173 58.3 B) PNPαGal WT 15.0 ± 2.0  2.51 ± 0.17 62.84.18 Δ2 13.3 ± 1.2  5.14 ± 0.82 128 9.65 Δ4 15.7 ± 1.0  5.74 ± 0.39 1439.13 Δ6 13.4 ± 1.1  1.89 ± 0.23 47.3 3.53 Δ8 13.0 ± 1.5  0.68 ± 0.0817.0 1.31 Δ10 17.0 ± 3.0  6.24 ± 0.12 156 9.18 The values given are forthe human enzyme purified from P. pastoris and assayed in triplicatefollowed by Lineweaver-Burk and non-linear regression analysis.Comparison of both Lineweaver-Burk and non-linear regression kineticparameters show good general agreement (data not shown). Non-linearregression results are displayed above. The k_(cat) was calculated using90 kDa as the MW of αGal. A) MUG was used as the substrate for enzymeassay. Mean and standard deviation measurements are from multiple assaysof three independent enzyme preparation for the Δ8 enzyme, twoindependent enzyme preparations for the WT enzyme, and single enzymepreparations for the other mutant enzymes. B) PNPαGal was used as thesubstrate for enzyme assay. Doi: 10.1371/journal.pone.0118341.t003

TABLE 4 Table 4. Purification on Table for WT αGal Expressed in P.pastoris. Specific Total Total Activity Purifi- Protein Activity (Units/cation Yield Step (mg) (Units × 10⁶) mg × 10³) (Fold) (%) Supernatant10,928 134 610 1.0 100 Con A Pool 138 30.4 221 18.1 22.8 Thio-Gal 4.1815.7 3,771 309 11.8 Pool Note. 5 mM MUG was used as the substrate forenzyme assay. Doi: 10.1371/journal.pone.011834.t004 PLOS ONE | DOI:10.1371/journal.pone.0118341 Feb. 26, 2015 10/26

TABLE 5 Table 5. Literature Values for K_(m) and V_(max) for the WTHuman αGal. K_(m) (mM) V_(max) (mmole/hr/mg) Source Reference Year 1.6NA Placenta [80] 1978 2.9 1.7 Liver [78] 1979 1.9 NA Plasma [60] 19792.5 NA Spleen [60] 1979 2.0 2.8 Spleen [69] 1981 2.3 2.3 Sf9 insectcells [81] 2000 2.0 4.8 Replagal [24] 2003 2.0 4.8 Fabrazyme [24] 20034.0 3.3 Fabrazyme [82] 2009 2.8 2.6 COS-7 cells [83] 2007 4.5 3.3 COS-7cells [84] 2011 Note. The values given are for the human enzyme purifieddirectly from human tissues or from the indicated recombinant sources.Replagal is produced in human foreskin fibroblasts and Fabrazyme isproduced in CHO cells. The average from these literature values are 2.6± 0.9 mM (K_(m)) and 3.2 ± 1.1 mmole/hr/mg (V_(max)). NA: not available.MUG was used as the substrate to determine the K_(m) and V_(max) values.Doi: 10.1371/journal.pone.0118341.t005

TABLE 6 Table 6. Literature Values of K_(m,) k_(cat,) and thespecificity constant (k_(cat)/K_(m)) for Glycosyl Hydrolase Family 27αGal Enzymes. Pubmed Accession Colloquial K_(m) k_(cat) k_(cat)/K_(m)Relative code Genus and species name (mM) (s⁻¹) (mM⁻¹s⁻¹) K_(cat)/K_(m)Ref. NP_000160 Homo sapiens Human 6.88 37.8 5.49 × 10³ 1 [95] NP_038491Mus musculus Mouse 1.40 N/A N/A N/A [96] WP_004844583.1 Ruminococcusgnavus Bacteria 1.80 30.1 1.67 × 10⁴ 3 [92] AAC99325 Saccharopolysporaerythraea Bacteria 0.650 23.3* 3.58 × 10⁴ 6 [98] P41947 Saccharomycescerevisiae Yeast 4.50 286 6.36 × 10⁴ 12 [99] BAB83765 Clostridium josuil(Catalytic Bacteria 0.810 61.9* 7.64 × 10⁴ 14 [100]  Domain) AAG24511Phanerochaete Fungus 0.198 272 1.37 × 10⁶ 250 [101]  chrysosporium Note.PNP αGal substrate was used to calculate kinetic values. Family 27enzymes include the human αGal and related enzymes in the CAZy database[102] that are most closely related as indicated by BLAST analysis [94].*k_(cat) values for S. erythraea and C. josuil were calculated based onthe reported V_(max), and molecular weights. Doi:10.1371/journal.pone.0118341.t006

TABLE 7 39.X, WT aGal from FR41 Aliquot# Contents Date Page# Vol (mL)U/mL Total Units mg/mL mg 39.1 Sup from FR41 7,000 72,825 509,775,00039.2 Sup after Centrifugation 26-Jan 5,500 91,575 503,662,500 39.3 Supafter 27-Jan αIII-240 438 304,800 133,502,400 25.0 10,935diafiltration + AC + buffer exchange to wash buffer 39.4 pool B2-E3 (FT24-Feb 1,100 20,280 22,308,000 load + wash ConA) 39.5 pool A4-F1 + G2-65(E1- 5-Mar αIII-243 1,800 26,640 47,952,000 E4, E6 ConA) 39.6 39.5 AC +buffer 5-Mar αIII-243 150 202,650 30,397,500 0.919 138 exchange tobinding 39.7 Thiogal Elution tube A6 15-Mar MMII70 14 95775 13408500.0504 0.706 39.8 Thiogal Elution tube A7 15-Mar MMII70 14 1097251536150 0.0323 0.452 39.9 Thiogal Elution tube A8 15-Mar MMII70 14118,725 1,662,150 0.0168 0.235 39.10 Thiogal Elution A5 29-Mar αIII-2434 110,100 440,400 0.0336 0.134 39.11 Thiogal Elution A9 29-Mar αIII-24314 101,475 1,420,650 0.0135 0.189 39.12 Thiogal Elution A10 29-MarαIII-243 14 104,250 1,459,500 0.0109 0.153 39.13 Thiogal Elution A1129-Mar αIII-243 14 2,823 39,517 39.14 Thiogal Elution A12 29-MarαIII-243 14 2,065 28,908 39.15 Thiogal pool Tubes A4-B12 240 65,6256,969,600 0.017 4.176 39.16 Thiogal Elution Tube A5 3-Apr αIII-2440.0470 2.780 0.131 2 mL----->47 uL 39.17 Thiogal Elution Tube A6 3-AprαIII-244 0.0410 3.530 0.145 1.5 mL---->41 uL 39.18 Thiogal Elution TubeA7 3-Apr αIII-244 0.0450 4.270 0.192 2 mL------>45 uL 39.19 ThiogalElution Tube A8 3-Apr αIII-244 0.0390 5.450 0.213 3.5 mL---->39 uL 39.20Thiogal pool 39.15 4-Apr αIII-244 60.0 277,050 16,623,000 0.0366 2.196AC + buffer exchange to 39.21 1700 uL of 39.20 -----> 5-Apr αIII-2440.0390 1.938 0.0756 39 uL of 39.21 39.22 500 uL of 39.6 ------> 5-AprαIII-244 0.0510 5.560 0.284 51 uL of 39.22 39.23 500 uL of 39.20 ----->2-May αIII-244 0.212 200 uL of 39.23 39.24 250 uL of 39.20 -----> 9-MayES219 0.070 70 uL of 39.24

The invention claimed is:
 1. A method of purifying recombinantalpha-galactosidase A, said method comprising: (a) obtaining asupernatant from cells recombinantly expressing alpha-galactosidase Agrown in a cell culture medium comprising non-precipitating phosphate;(b) contacting said supernatant with a chromatography media, whereinsaid chromatography media comprises media that binds α-D-mannopyranosylor α-D-glucopyranosyl; and (c) eluting alpha-galactosidase A from saidchromatography media to generate an eluate comprisingalpha-galactosidase A, wherein said eluting comprises at least oneelution pause between 4 and 16 hours.
 2. The method of claim 1, whereinsaid supernatant is clarified prior to contacting the chromatographymedia.
 3. The method of claim 1, wherein said non-precipitatingphosphate comprises sodium hexametaphosphate.
 4. The method of claim 1,wherein said chromatography media comprises Concanavalin A.
 5. Themethod of claim 1, wherein said eluate is contacted with achromatography media comprising D-galactose.
 6. The method of claim 1,wherein said elution pause is between 10 and 14 hours.
 7. The method ofclaim 1, wherein the eluate is subject to diafiltration.
 8. The methodof claim 1, wherein the eluate is subject to buffer exchange.
 9. Themethod of claim 1, wherein the alpha-galactosidase A is truncated.